The present invention is directed to a novel arginine deiminase and long-acting arginine deiminase-containing compositions. In particular the invention is directed to substantially non-antigenic polymer conjugates containing arginine deiminase derived from Mycoplasma arthritidis which demonstrate high levels of retained enzyme activity.
Conjugating biologically-active proteins or enzymes to polymers has been suggested to improve one or more of the properties of circulating life, water solubility or antigenicity in vivo. For example, some of the initial concepts of coupling peptides or polypeptides to polyethylene glycol (PEG) and similar water-soluble polymers are disclosed in U.S. Pat. No. 4,179,337, the disclosure of which is incorporated herein by reference. Conjugates are formed by reacting a biologically active material with a several fold molar excess of a polymer which has been modified to contain a terminal linking group. Insulin and hemoglobin were among the first therapeutic agents conjugated. These relatively large polypeptides contain several free .epsilon.-amino attachment sites. Several polymers could be attached without significant loss of biologic activity. The conjugation process, however, is not without complications. Excessive polymer conjugation or reactions using molar excesses of polymers beyond certain ratios can result in the formation of inactive conjugates or conjugates having insufficient activity. Problems often result when the active site (i.e. where groups associated with bioactivity are found) on the protein or enzyme becomes blocked as a result of the covalent polymer attachment. This problem can be difficult to avoid because the polymer and protein or enzyme are typically joined in solution-based reactions. Pre-blocking the active sites with reversible materials such as pyridoxal phosphate has been suggested, but the results have been inconsistent. The problems are particularly acute with relatively lower molecular weight proteins and peptides. These bioactive materials often have few attachment sites not associated with bioactivity.
Arginine deiminase (ADI) is one type of enzyme which could benefit from improved polymer conjugation techniques. ADI is an enzyme that hydrolyzes arginine and the depletion of arginine has been postulated to have a killing effect on certain tumors. In particular, the enzyme hydrolyzes the guanidino group of L-arginine into L-citrulline and ammonia. The ADI enzyme is also known as arginine dihydrolase, arginine desiminase or guanidinodesiminase. Although ADI has been obtained from other types of mycoplasma strains as well as other microorganism sources such as pseudomonas and streptococcus, there is variability in the enzyme obtained. In particular, each host strain produces an enzyme having a different number of lysines as well as variations in the active site to produce an enzyme having a different primary sequence, and number and location of lysines.
Several polymer-arginine deiminase conjugates have previously been suggested. See, for example, Jpn. J. Cancer Res. 84, 1195-1200, November 1993 which describes inter alia arginine deiminase from Mycoplasma arginini conjugated with methoxy-polyethylene glycol4,6-dichloro-1,3,5-triazine. The previous arginine deiminase-polymer conjugates prepared to date, however, have been deemed to be unacceptable. One of the chief drawbacks has been that the level of retained arginine deiminase activity provided by highly modified conjugates has been too low in growth inhibition studies. It has been postulated that certain lysine attachment points on the enzyme are intimately connected with the enzyme active site. Therefore, the highly modified conjugates which demonstrate high levels of retained activity were not possible at reasonable expenditures of time and resources.
The present invention addresses these shortcomings.